The following discussion describes certain art references, none of which is admitted to be prior art to the invention described herein.
Methylthioadenosine phosphorylase (MTAP) is an enzyme found in all normal tissues that catalyzes the conversion of methylthioadenosine (MTA) into adenine and 5-methylthioribose-1-phosphate. Afterward, the adenine is salvaged to generate adenosine monophosphate, and the 5-methylthioribose-1-phosphate is converted to methionine and formate. Because of this salvage pathway, MTA can serve as an alternative purine source if de novo purine synthesis is blocked, e.g., with antimetabolites, such as L-alanosine.
Many human and murine malignant cells lack MTAP activity. MTAP deficiency is not only found in tissue culture cells but the deficiency is also present in primary leukemias, gliomas, melanomas, pancreatic cancers, non-small cell lung cancers (NSLC), bladder cancers, astrocytomas, osteosarcomas, head and neck cancers, myxoid chondrosarcomas, ovarian cancers, endometirial cancers, breast cancers, soft tissue sarcomas, non-Hodgkin lymphomas, and mesotheliomas (Kamatani et al. (1981) Proc. Natl. Acad. Sci USA 78:1219–1223; Toohey (1977) Biochem. Biophys. Res. Commun. 78:1273–1280; Fitchen et al. (1986) Cancer Res. 46:5409–5412; Nobori et al. (1991) Cancer Res. 51:3193–3197; Nobori et al. (1993) Cancer Res. 53:1098–1101; Christopher et al. (2002) Cancer Res. 62:6639–6644; and Garcia-Castellano et al. (2002) Clin. Cancer. Res. 8:782–787). Homozygous deletion is frequently the mechanism for inactivation of the gene encoding MTAP (Christopher et al. (2002) Cancer Research 62:6639–6644). Other mechanisms for MTAP deficiency, however, have been reported (Harasawa et al. (2002) Leukemia 16:1799–1807).
The gene encoding for human MTAP maps to region 9p21 on human chromosome 9p. This region also contains the tumor suppressor genes p16INK4A (also know as CDKN2A), and p15INK4B. These genes encode for p16 and p15, which are inhibitors of the cyclin D-dependent kinases cdk4 and cdk6, respectively (Efferth et al. (2002) Blood, Cells, Molec., and Dis. 28:47–56; Kamp et al.(1994) Science 264:436–440; Hannon et al. (1994), Nature 371:257–261). The p16INK4A transcript can be alternatively ARF ARF spliced into a transcript encoding p14ARF. p14ARF binds to MDM2 and prevents degradation of p53 (Pomerantz et al. (1998) Cell 92:713–723).
The 9p21 chromosomal region is of interest because it is frequently homozygously deleted in a variety of cancers, including leukemias, NSLC, pancreatic cancers, gliomas, melanomas, and mesothelioma. The deletions often inactivate more than one gene. For example, Cairns et al. ((1995) Nat. Gen. 11:210–212) reported that after studying more than 500 primary tumors, almost all the deletions identified in such tumors involved a 170 kb region containing MTAP, p14ARF and P16INK4A Carson et al. (WO 99/67634) reported that a correlation exists between the stage of tumor development and loss of homozygosity of the gene encoding MTAP and the gene encoding p16. For example, deletion of the MTAP gene, but not p16INK4A was reported to be indicative of a cancer at an early stage of development, whereas deletion of the genes encoding for p16 and MTAP was reported to be indicative of a cancer at a more advanced stage of tumor development. Garcia-Castellano et al. reported that in some osteosarcoma patients, the MTAP gene was present at diagnosis but was deleted at a later time point (Garcia-Castellano et al., supra).
Reference protein sequences for p16 and alternative transcripts, including p14, are deposited in GenBank under the following accession numbers NP—000068; NP—478102.1; NP—478103.1, and NP—478104.1. Reference mRNA sequences for p16 and alternative transcripts, including p14, are deposited in Genbank under accession numbers NM 000077.2; NM—058195.1; NM 058196.1; and NM—058197.1. Reference protein sequences for p15 are deposited under GenBank accession numbers NP—004927.2 and NP—511042.1. Reference mRNA sequences for p15 are deposited in GenBank under accession numbers NM—004936.2 and NM—078487.1.
Methods for determining the MTAP status in tumor cells have been described. U.S. Pat. No. 5,942,393 is said to describe methods for detecting MTAP-encoding nucleic acid through use of oligonucleotide probes. Norbori et al. ((1991) Cancer Res. 51:3193–3197); and (1993) Cancer Res. 53:1098–1101) reported the use of a polyclonal antisera to bovine MTAP to detect MTAP protein isolated from tumor cell lines or primary tumor specimens in an immunoblot analysis. Garcia-Castellano et al. (2002, supra) report the use of antihuman MTAP chicken antibody to screen osteosarcoma tumor samples that were embedded in OCT frozen blocks.
Because many tumor cells are MTAP deficient and, therefore, dependent on de novo purine synthesis for growth and/or survival, the MTAP salvage pathway may offer an opportunity for selective tumor therapy which spares normal tissues. To this end, the development of therapies based on treating MTAP deficient cancers with chemotherapeutic regimens that interfere with purine utilization is presently underway. Thus, a need exists for compositions and methods that identify MTAP deficient tumors.
In particular, a need exists for compositions and methods that identify MTAP deficient tumors (tumor cells that produce no or low amounts of MTAP protein) in biological samples, particularly those biological samples commonly used in a medical environment, such as formalin-fixed paraffin-embedded (FFPE) tissue specimens. The ability to detect human MTAP protein using immunohistochemistry techniques may be advantageous over other immunoassay techniques, such as Western blotting, as well as oligonucleotide based procedures, such as Southern blotting, in that individual cells can be screened and the chance of contaminating tumor cells with normal cells is reduced.